Suppression of nonsense mutations as a therapeutic approach to

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UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
Suppression of nonsense mutations as a therapeutic
approach to treat genetic diseases
Francesca Manuela Johnson de Sousa Brito
DISSERTAÇÃO
MESTRADO EM BIOLOGIA HUMANA E AMBIENTE
2014
UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
Suppression of nonsense mutations as a therapeutic
approach to treat genetic diseases
Dissertation oriented by:
Doutora Luísa Romão
(Instituto Nacional de Saúde Dr. Ricardo Jorge)
Prof. Doutora Ana Crespo
(Departamento de Biologia Animal, Faculdade de Ciências da Universidade de Lisboa)
Francesca Manuela Johnson de Sousa Brito
MESTRADO EM BIOLOGIA HUMANA E AMBIENTE
2014
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iii
“If I had a world of my own, everything would be nonsense. Nothing would be what it
is, because everything would be what it isn't. And contrary wise, what is, it wouldn't
be. And what it wouldn't be, it would. You see?”
Lewis Carroll
(English writer, 1832-1898)
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v
Acknowledgments
Though only my name appears on the cover of this dissertation, a great deal of people
have contributed to its production and whom without I would have never gotten this
far and been able to complete this thesis. I owe my gratitude to all those people who
have made this dissertation possible and because of whom my experience has been
one that I will cherish forever.
First and foremost, I would like to express my sincere gratitude to my advisor Dr. Luísa
Romão for accepting me in her research group. For her patience, motivation,
enthusiasm, guidance, immense knowledge, and allowing me the room to work in my
own way I am extremely grateful.
I would like to thank Prof. Ana Crespo, my co-advisor, for all her teachings, for her
support and interest in the development of this thesis.
I also want to thank to Dr. João Lavinha for allowing me to carry out this study at
Departamento de Genética Humana from Instituto Nacional de Saúde Dr. Ricardo
Jorge.
I have been blessed with the people with whom I shared my daily work. To Alexandre
Teixeira, thank you for teaching me how to think as a true scientist, or at least try to.
Even though he sometimes was a true pain I am truly grateful for all the things he
taught me and for all the time he dedicated to me even when he had no time to spare.
He wasn’t just a teacher to me but also a friend that was always available to give me
good advice. I hope this thesis will answer his daily question to me throughout this
project, “what is your project?”, if not at least I tried. I wish for him all the luck in the
universe for this new chapter of his life. To Rafaela, I am indebted to her. As well as
teaching me all I needed to know to be able to work in a genetics lab and do my
experiments for this project, she has guided me and has been my friend, and what a
good and true friend she has become. She changed the place where I worked into
something more than just a workplace and I want to thank her so much for that. I am
thankful for all our scientific and non scientific discussions which helped me discuss my
results in this dissertation. Thank you also for patiently reading and correcting my
thesis, over and over and over and over again. And last but not least, I am also thankful
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for all the wonderful deserts she brought to the lab on occasion, they made those long
days a little less harder.
Claudia Onofre, I thank her for letting me steal her primers, ethanol and anything else I
could get my hands on. She might not think it but she has taught me a lot during this
past year and has been a good friend when needed. It has been great fun planning our
imaginary weddings together. Ana Ramos, Juliane Menezes and Bruno Silva, it has
been a pleasure sharing my days with them. They have made them fun and brightened
many a dull lunchtimes and any other break during our days at the lab. Paulo Costa,
even though he has not been with us for long he never fails to amuse us with his dry
wit all throughout the process of writing my thesis.
I would also like to thank my university Professor, Patricia Rodrigues. She has not just
been a mentor to me but a friend, helping me throughout all my decision making and
guiding me throughout this entire journey. I thank her for giving me the fantastic
opportunity of being able to shadow her and for all the wonderful things she has
taught me.
To my dearest friend, Ana Luísa, who is always there when I need her, I’m thankful for
her support and most of all for her friendship. Even far away from each other and only
seeing each other once in a while, when we do see each other we pick up just were we
left off. After all, that’s what best friends do. We will always be friends until we are old
and senile, and then we will be new friends.
I would like to thank my boyfriend, André, for his love and support, for taking me to
work every day even though I know deep down he wished he was still in bed sleeping. I
thank him for driving me through all the wonderful streets of Lisbon at night listening
to Smooth.fm just so I would relax a bit after all the long hours writing this
dissertation. I know that these past few months haven’t been easy, but I am grateful
that he has stood by my side through this journey.
Most importantly, none of this would have been possible without the love and
patience of my family. To my sister Jessica, the smartest teenager I know, I’m thankful
for all the times she came to visit me in Lisbon, for her silly faces and dances when I
went home, and for all the times she has made me laugh when I most needed to. To
my parents, I thank them for making me the person I am today, for having faith in me
and for their unconditional love and support, both financially and emotionally
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throughout my life. Without them I wouldn’t have gotten where I am today. I thank
them for providing me with a beautiful home away from home and for always
receiving me with arms wide open each time I went home. I hope this thesis makes
you proud as I dedicate it to you.
viii
Abstract
Premature termination codons (PTCs or nonsense codons) can arise from various types
of mutations in germ or somatic cells. PTCs promote premature translational
termination and the induction of nonsense-mediated mRNA decay (NMD). NMD is a
surveillance system that prevents the synthesis of C-terminally truncated proteins toxic
for the cell. The physiological importance of NMD is manifested by the fact that about
one third of genetic disease-associated mutations generate PTCs.
In recent years, a novel therapeutic approach called suppression therapy has been
developed that utilizes low molecular weight compounds to induce the translation
machinery to recode a PTC into a sense codon. Beta-thalassaemia (β-thalassaemia) is
one of the most common genetic diseases worldwide, and nonsense mutations
occurring in the beta-globin (β-globin) are among the most frequent mutations
associated with β-thalassaemia. Some studies have shown that aminoglycosides, low
molecular weight compounds, and non-aminoglycosides can suppress PTCs in cystic
fibrosis and Duchenne’s muscular dystrophy, but it remains unclear whether βthalassaemia would also be responsive to a similar drug treatment. Preliminary results
obtained in our lab have shown that the aminoglycoside G418 can suppress a
nonsense mutation at codon 39 of the human β-globin mRNA, although at low levels in
cultured erythroid cells. To investigate if suppression therapy can restore enough βglobin protein to correct the disease manifestations of β-thalassaemia, we tested
whether G418 is able to induce efficient levels of suppression in a dose-dependent and
time-course manner in HeLa cells transfected with plasmids containing the human βglobin wild type gene (βWT) or the counterpart carrying a nonsense mutation at codon
39 (β39).
Our results show a slight increase in the levels of mRNA of the β39 transcript which
may indicate a possible effect by the drug. Seen as we were unable to test the effect of
the drug on a protein level, in the future, it would be of great interest to evaluate its
effect on protein expression levels.
Key-words: Nonsense-mediated mRNA decay (NMD); mRNA translation; genetic
diseases; suppression therapy; G418.
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Sumário
Algumas das mutações que ocorrem em células somáticas ou na linha germinal podem
resultar na formação de codões de terminação prematura da tradução (CTPs ou
codões nonsense). Um terço das mutações associadas a doenças genéticas resulta na
produção de CTPs. Em geral, a introdução dum CTP num transcrito induz a terminação
prematura da tradução e a activação do mecanismo de decaimento do mRNA mediado
por mutações nonsense (nonsense-mediated mRNA decay; NMD). O NMD é um
mecanismo de controlo de qualidade que impede a produção de proteínas truncadas
na extremidade C-terminal tóxicas para a célula.
No decorrer dos últimos anos, tem sido desenvolvida uma nova terapia, designada
como terapia de supressão, que utiliza compostos de baixo peso molecular para
induzir a maquinaria de tradução a alterar um CTP para um codão sense. O objectivo
da terapia de supressão é estimular a capacidade de competição de tRNAs aminoacil
com o complexo de terminação para a ligação ao CTP no local A do ribossoma, de
forma a suprimir especificamente a terminação em CTPs e não em codões de
terminação naturais. Embora a talassémia seja uma das doenças genéticas mais
comuns e as mutações nonsense no gene da beta-globina (β-globina) estejam muito
frequentemente associadas à beta-talassémia (β-talassémia), a utilização da terapia de
supressão nestes casos ainda não foi investigada de forma detalhada. Resultados
preliminares obtidos pelo nosso laboratório indicam que o aminoglicósido G418 tem a
capacidade de suprimir uma mutação nonsense no codão 39 do mRNA da β-globina,
em células eritróides em cultura. De forma a confirmar o princípio de que a terapia de
supressão pode restaurar os níveis de β-globina, testámos se o composto G418 tem a
capacidade de induzir níveis significativos de supressão em células HeLa transfectadas
com plasmídeos que contêm o gene da β-globina humana normal (βWT) ou o
equivalente com mutação nonsense no codão 39 (β39).
Segundo os resultados obtidos, há um ligeiro aumento nos níveis de transcrito β39 o
que pode ser indicador de um possível efeito da droga. Como não nos foi possível
verificar o efeito da droga nos níveis de proteína é necessário trabalho futuro para
avaliar o seu efeito nos níveis de expressão proteica.
Palavras-chave: Decaimento do mRNA mediado por mutações nonsense (NMD);
tradução do mRNA; doenças genéticas; terapia de supressão; G418.
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Abbreviations
A
Adenine
A-site
aminoacyl-site
bp
base pairs
CBC
cap binding complex
CBP
cap binding protein
cDNA
complementary deoxyribonucleic acid
C-terminal
carboxyl-terminal
DEAD
Asp-Glu-Ala-Asp motif
DECID
decay inducing complex
DH5α
strain of Escherichia coli
DMEM
Dulbecco's modified Eagle medium
DNA
deoxyribonucleic acid
eEF
eukaryotic translation elongation factor
eIF
eukaryotic initiation factor
EJC
exon junction complex
eRF
eukaryotic translation release factor
E-site
exit-site
FBS
foetal bovine serum
G
guanine
GDP
guanosine diphosphate
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GTP
guanosine triphosphate
h
hour(s)
HeLa
human cervical cancer cell line
IRES
internal ribosome entry site
MAGOH
mago-nashi homolog
Met
metionin
Met-tRNAi
methionine-loaded initiator tRNA
min
minute(s)
mRNA
messenger ribonucleic acid
mRNP
messenger ribonucleoprotein particle
nt
(any) nucleotide
N-terminal
amino-terminus
NMD
nonsense-mediated mRNA decay
ORF
open reading frame
PABP
poly(A)-binding protein
PABPC1
cytoplasmic poly(A)-binding protein 1
PAN
poly(A) nuclease
PBS
phosphate buffered saline
PIN
PilT N terminus
PCR
polymerase chain reaction
PLB
passive lysis buffer
Poly(A)
poly-adenilate
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Pre-mRNA
messenger ribonucleic acid precursor
P-site
peptidyl-site
PTC
premature translation termination codon
Puro
puromycin
RNA
ribonucleic acid
RNase
ribonuclease
RNPS1
RNA binding protein S1
RT
reverse transcription
RT-qPCR
reverse transcription quantitative polymerase chain reaction
sec
second(s)
SMG
suppressor of morphological defects on genitalia
Srm160
SR-related nuclear matrix protein of 160 kDa
SURF
SMG1-UPF1-eRFs complex
T
thymine
tRNA
transfer ribonucleic acid
tRNAi
transfer ribonucleic acid of initiation
U
uracil
uORF
upstream open reading frame
UPF
up-frameshift protein
UTR
untranslated region
WT
wild type
XRN1
5’-3’ exoribonuclease 1
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β15
human β‐globin transcript with a PTC at codon 15
β39
human β‐globin transcript with a PTC at codon 39
βWT
normal/wild type human β‐globin transcript
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Index
I. Introduction ...................................................................................................................1
I.1. mRNA translation.......................................................................................................2
I.1.1. Translation Initiation...................................................................................2
I.1.2. Translation Elongation................................................................................4
I.1.3. Translation Termination..............................................................................6
I.2. Nonsense-mediated mRNA decay- a surveillance mechanism...................................7
I.2.1. PTC recognition...........................................................................................7
I.2.2. Molecular events that prepare mRNA for degradation by NMD................8
I.2.3 NMD degradation mechanism...................................................................10
I.2.4. NMD targets..............................................................................................13
I.2.5. Implication of NMD in disease..................................................................13
I.2.6.β-Thalassaemia as a model disease for studying NMD.............................15
I.2.7. Suppression therapy.................................................................................16
I.3. Aims..........................................................................................................................18
II. Methods.................................................................................................................................18
II.1 Plasmid constructs....................................................................................................18
II.2 Cell culture and plasmid transfections.....................................................................19
II.3 RNA isolation............................................................................................................19
II.4 Reverse transcription-coupled quantitative PCR (RT-qPCR).....................................19
II.5. SDS-PAGE and Western blotting..............................................................................20
II.6. Statistical analysis....................................................................................................20
III. Results and Discussion........................................................................................................21
III.1. Sequence analysis of the plasmids DNA encoding the β-globin gene to confirm the
wild type sequence and the presence of mutations at codons 15 and 39.....................21
III.2. Analysis of βWT, β15 and β39 transcripts in transiently transfected HeLa
cells.................................................................................................................................22
III.2.1. mRNA quantification by RT-qPCR analysis..............................................23
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III.2.2. Protein analysis by Western Blot............................................................24
III.3. Analysis of βWT and β39 transcripts in transiently transfected HeLa cells exposed
to geneticin (G418).........................................................................................................25
IV. Future directions.................................................................................................................27
V. References.............................................................................................................................29
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I. Introduction
Eukaryotic gene expression pathway involves a series of interconnected steps, from
transcription to translation. These steps are integrated with one another to augment
the efficiency and fidelity of gene expression. This allows expression of individual
genes to be controlled, producing transcripts and eventually detecting and degrading
aberrant transcripts (Chang et al, 2007; Clancy & Brown, 2008; Nicholson et al, 2010;
Behm-Ansmant et al, 2007). If not detected and degraded, these transcripts can result
in the accumulation of potentially harmful truncated proteins (Nicholson et al, 2010).
Given the complex chain of biochemical reactions involved in transforming the genetic
information of an organism into gene products, the overall accuracy of gene
expression is quite astonishing (Mühlemann & Lykke-Andersen, 2010). Since mRNAs
function primarily as templates for protein synthesis, it is now established that most, if
not all, of the steps of the gene expression pathway can be regulated by qualitycontrol mechanisms (or mRNA surveillance mechanisms) both in the nucleus and in the
cytoplasm at multiple stages during gene expression. For example, improperly
processed mRNAs are degraded by mRNA surveillance mechanisms in the nucleus
before they are exported. In the cytoplasm, quality control mechanisms assess the
translatability of the mRNA and degrade any that lacks translation termination codons
or that has premature translation termination codons (PTCs), thereby preventing the
accumulation of potentially toxic protein fragments (Schoenberg & Maquat, 2012;
Behm-Ansmant et al, 2007; Nicholson et al, 2010). Nonsense-mediated mRNA decay
(NMD) represents a translation-dependent post-transcriptional process that selectively
recognizes and degrades mRNAs whose open reading frame (ORF) is truncated by a
PTC. In doing so, NMD protects the cell from accumulating C-terminally truncated
proteins with potentially deleterious functions (Mühlemann et al, 2008).
NMD acts not only on aberrant mRNAs, but also regulates the expression of naturally
occurring transcripts having features that allow them to be recognized as PTCcontaining transcripts. In this way, NMD also contributes to the post-transcriptional
regulation of gene expression (Behm-Ansmant et al, 2007).
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I.1. mRNA translation
In order to understand how and when the surveillance mechanisms can act, we need
to understand the pathway of gene expression in further detail. One of the most
important steps in this pathway is the translation of proteins from messenger RNA
(mRNA). Regulation of translation is a mechanism that is used to modulate gene
expression in a wide range of biological situations. From early embryonic development
to cell differentiation and metabolism, translation regulation is used to fine-tune
protein levels in both time and space (Gebauer & Hentze, 2004).
During translation, the sequence of codons on mRNA directs the synthesis of a
polypeptide chain. This dynamic process is usually divided into three phases: initiation,
elongation and termination (Preiss & Hentze, 2003; Ramakrishnan, 2002). Ribosomes,
large ribonucleoprotein particles composed of two subunits in all species, are, along
with numerous translation factors, the cellular machines responsible for translating
genetic information into polypeptide sequences. During the step-wise movement of an
mRNA through the ribosome, amino acids are incorporated into the elongating
polypeptide chain. Each subunit has three binding sites for tRNA, designated: the A
(aminoacyl), which accepts the incoming aminoacylated tRNA; P (peptidyl), which
holds the tRNA with the nascent peptide chain; and E (exit), which holds the
deacylated tRNA before it leaves the ribosome. The fidelity of amino acids
incorporation depends on base-pair complementarities between sense codons and
anticodons of aminoacyl-tRNAs (Korostelev, 2011; Ramakrishnan, 2002). We will
discuss the three phases of translation in the following paragraphs.
I.1.1.Translation initiation
The translation of mRNA begins with the formation of the ternary complex composed
of eukaryotic initiation factor 2 (eIF2), a hetero-dimer of 3 subunits (α, β and γ), bound
to the methionyl-initiator tRNA (Met-tRNAiMet), and GTP by the γ subunit (fig 1. A)
(Gebauer & Hentze, 2004). Once the ternary complex is assembled and active, it must
bind to the 40S ribosomal subunit. This binding is aided by eukaryotic initiation factors
1, 1A, 3 and 5. The structure resulting from the bound of ternary complex to 40S
2
ribosomal subunit alongside initiation factors 1, 1A, 3 and 5 is designated 43S preinitiation complex (fig 1. B) (Gebauer & Hentze, 2004; Jackson et al, 2010).
A
B
C
D
E
Figure 1: Cap-mediated translation initiation. The methionine-loaded initiator tRNA binds to
GTP-coupled eIF2, to yield the ternary complex. This complex then binds to the small (40S)
ribosomal subunit, eIF3 and other initiation factors to form the 43S pre-initiation complex. The
pre-initiation complex recognizes the mRNA by the binding of eIF3 to the eIF4G subunit of the
cap-binding complex. In addition to eIF4G, the cap-binding complex contains eIF4E, which
directly binds to the cap, and eIF4A, an RNA helicase that unwinds secondary structure during
the subsequent step of scanning. The 43S pre-initiation complex scans the mRNA in a 5′ to 3′
direction until it identifies the initiator codon AUG. Scanning is assisted by the factors eIF1 and
eIF1A. Stable binding of the 43S pre-initiation complex to the AUG codon yields the 48S
initiation complex. Subsequent joining of the large (60S) ribosomal subunit results in the
formation of the 80S initiation complex. Both AUG recognition and joining of the large
ribosomal subunit trigger GTP hydrolysis on eIF2 and eIF5B, respectively. Subsequently, the
80S complex is competent to catalyze the formation of the first peptide bond. Pi: inorganic
phosphate (Gebauer & Hentze, 2004).
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The 5’ cap-proximal region is recognized by eIF4F. This complex comprises the capbinding protein eIF4E; the DEAD-box RNA helicase eIF4A, which unwinds secondary
structures in the 5’ untranslated region (5’UTR) so that the 43S complex can bind and
scan the mRNA; and eIF4G, which functions as a ‘scaffold’ that binds eIF4E and eIF4A in
order to form eIF4F, the cap-binding complex of translation initiation. The 43S complex
then scans the 5′UTR in the 5′ to 3′ direction, downstream of the cap, until it finds the
first AUG in good initiation context (fig 1. C) (scanning model of translation initiation;
Kozak, 1989). The 43S complex recognizes the initiation codon through the formation
of base pairs between the initiator tRNA and the start codon and formation of 48S
initiation complex occurs (Jackson et al, 2010; Gebauer & Hentze, 2004). After 48S
complex formation and initiation codon recognition, eIF5 promotes the hydrolysis of
eIF2-bound GTP and eIF5B, a ribosome-dependent GTPase, and the displacement of
eIFs and the joining of the 60S subunit (fig 1. D), leading to the assembly of 80S
ribosome (fig 1.E), which is competent to initiate elongation. Although this is the
canonical mechanism by which protein synthesis in eukaryotes occurs, there are some
alternatives to this translation initiation model which may be cap-dependent (e.g.
leaky scanning, reinitiation) or independent such as translation mediated by internal
ribosome entry sites (IRESs) or CITEs among some other mechanisms that explain
exceptions to this rule (Jackson et al, 2010; Gebauer & Hentze, 2004).
I.1.2. Translation Elongation
The end of the initiation process leaves an aminoacylated initiator tRNA in the P site of
the ribosome and an empty A site, which serves to start the elongation cycle
(Ramakrishnan, 2002). The elongation phase of protein synthesis involves the correct
decoding of the mRNA into the amino acid sequence of the encoded polypeptide,
involving fewer factors than initiation. A major feature of elongation must therefore be
to maintain the accuracy of the process, to ensure errors are not made in synthesising
the product (Proud, 1994).
During elongation, amino acids are added sequentially to the growing polypeptide
chain, in the order specified by the sequence of the mRNA in frame with the AUG
codon. A key factor in this process is eukaryotic elongation factor eEF1A, which is
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responsible for delivering the aminoacyl-tRNA to the A-site of the ribosome. The
activity of eEF1A is dependent on GTP; the guanine nucleotide-exchange factor eEF1B
promotes the regeneration of active eEF1A–GTP complexes. The last elongation factor
is eEF2, which is required for the translocation of the peptidyl-tRNA from the A-site of
the ribosome to the P-site and the movement of the ribosome along the mRNA. This is
also a GTP-dependent process (fig 2) (Abbot & Proud, 2004).
Figure 2: Model of the eukaryotic translation elongation pathway. Starting at the top, an
eEF1A-GTP-aminoacyl-tRNA ternary complex binds the aminoacyl-tRNA to the 80S ribosome
with the anticodon loop of the tRNA in contact with the mRNA in the A-site of the small
subunit. Following release of eEF1A-GDP, the aminoacyl-tRNA is accommodated into the A
site, and the eEF1A-GDP is recycled to eEF1A-GTP by the exchange factor eEF1B. Peptide bond
formation is accompanied by transition of the A- and P-site tRNAs into hybrid states with the
acceptors ends of the tRNAs moving to the P and E sites, respectively. Binding of eEF2-GTP
promotes translocation of the tRNAs into the P and E sites, and is followed by release of eEF2GDP. The ribosome is now ready for the next cycle of elongation with release of the deacylated
tRNA from the E site and binding of the appropriate eEF1A-GTP.aminoacyl-tRNAto the A-site.
GTP: depicted as a green ball; GDP: depicted as a red ball (Dever & Green, 2012)
The process of elongation is repeated many times, until a stop codon is reached. An
additional amino acid is added to the growing polypeptide chain each time the mRNA
advances through the ribosome. Once a polypeptide chain of reasonable size is
assembled, it begins to emerge from the base of the large subunit (Klug et al, 2005).
5
When a stop codon is recognized by the ribosome, translation terminates, and the
nascent protein must be released from the mRNA and ribosome (Clancy & Brown,
2008).
I.1.3. Translation Termination
The final step of translation - termination - takes place when a stop codon, UAG, UAA,
or UGA, enters the A-site of the ribosome. For simplicity, termination can be thought
of as two distinct steps, stop codon recognition and peptide release. In eukaryotes,
translation termination is mediated by eukaryotic release factor 1 (eRF1), which is
responsible for stop codon recognition and triggering peptide release, and eRF3, a
GTPase that stimulates eRF1-mediated peptide release (Abbot & Proud, 2004). eRF1, in
turn, stabilizes binding of GTP to eRF3 so that they form a stable ternary complex
(Mitkevich et al, 2006; Pisareva et al, 2006). The binding of the eRF complex to the
ribosome stimulates the cleavage of the bond between the peptide and the tRNA,
thus, releasing the newly synthesized peptide and the tRNA from the ribosome, which
then dissociates into its subunits. If a stop codon should appear in the middle of an
mRNA molecule, the same process occurs, and the polypeptide chain is prematurely
terminated (Abbot & Proud, 2004; Klug et al, 2005; Dever & Green, 2012; Karijolich &
Yu, 2014). Interactions of the eRFs with cellular proteins playing key roles in other gene
expression processes may be the reason by which termination activity is adjusted and
linked to other events in mRNA translation and NMD (fig 3) (Dever & Green, 2012).
Figure 3: Model of eukaryotic translation termination. A complex comprised of eRF1 and eRF3
mediate translation termination. eRF1 recognizes any of the three stop codons (UAA, UAG,
UGA) in the ribosomal A site. GTP hydrolysis by eRF3 assists: 1) stop codon recognition by
eRF1, and 2) eRF1 accommodation into the peptidyl transferase center so polypeptide release
can occur (Keeling et al, 2012).
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I.2. Nonsense-mediated mRNA decay - a surveillance mechanism
At the level of mRNA, two important features are monitored by cell quality control
mechanisms: first, whether it has the correct set of proteins bound to a particular
mRNA; and, secondly, whether the coding potential of the mRNA is intact (Nicholson &
Mühlemann, 2010).
There are several quality control mechanisms in mammalian cells such as nonsensemediated mRNA decay (NMD), no-go mRNA decay and nonstop mRNA decay. Here, we
are going to focus on NMD seen as it is the best characterized of the three and the only
one studied in any detail from a regulatory perspective (Schoenberg & Maquat, 2012).
NMD is one of the best characterized eukaryotic mRNA quality control mechanisms.
NMD targets mRNAs harbouring premature termination (nonsense) codons (PTCs) for
degradation. This pathway is important because if PTC-containing messages were
allowed to be translated they would produce potentially toxic truncated proteins with
potentially deleterious gain-of-function or dominant-negative activity (Nicholson &
Mühlemann, 2010; Behm-Ansmant et al, 2007; Chang et al, 2007; Nicholson et al,
2010).
All organisms require the NMD pathway to eliminate transcripts containing PTCs,
which arise from inherited or sporadic mutations or alternative splicing, so that normal
cellular function is maintained; and to eliminate endogenous error-free transcripts in
order to maintain regular levels of transcript and subsequent protein synthesis for
development and viability (Chapin et al, 2014).
I.2.1. PTC recognition
Even before the identification of the NMD molecular players, a rule for the recognition
of PTCs that induce mammalian NMD was postulated. Studies in mammalian systems
led to the observation that PTCs, when located more than 50-54 nucleotides upstream
the last exon-exon junction, were able to target mRNA for decay, whereas PTCs
located downstream of this boundary do not induce NMD. The discovery of the exon
junction complex provided a molecular explanation for the empirically detected ’50-54
nucleotide boundary’ and supports the view that PTC recognition is dependent on the
7
definition of the exon-exon junctions, suggesting that the process of splicing is
implicated in mammalian NMD (Silva & Romão, 2009; Schweingruber et al, 2013).
A major issue that is still only partially understood is how NMD machinery
distinguishes between premature and normal stop codons. Generation of a PTC can
involve as little as a single nucleotide change. How such a subtle alteration can be
detected as aberrant has led to several models. In all models, detection is intimately
connected to translation termination. Synthesized mRNAs are bound by the capbinding protein heterodimer CBP80-CBP20, which constitutes the cap-binding complex
(CBC), and if derived from intron-containing pre-mRNA, they are also bound by
multiprotein assemblies, the EJCs, that presently are known to assemble ~20-24 nts
upstream of each exon-exon junction during pre-mRNA splicing. The EJC contains the
general splicing activator RNPS1, the RNA export factor Aly/ REF, the shuttling protein
Y14, the nuclear matrix-localized serine-arginine-containing protein SRm160, the
oncoprotein DEK, and the Y14 binding protein magoh. The interaction of magoh with
Y14 may have a role in anchoring the NMD-specific factors UPF3 and UPF2 to the
mRNA. Translation termination, which involves eRF1 and eRF3, provides the first signal
necessary for activation of NMD. According to the present models, translating
ribosomes displace EJCs from the open reading frame (ORF) during the “pioneer”
round of translation. If assembly of eRF1-eRF3 at a termination codon occurs ≥ 50-54
nucleotides (nts) upstream from an exon-exon junction, the footprint of the
terminating ribosome is insufficient to physically remove the EJCs (Chang et al, 2007;
Popp & Maquat, 2013; Inácio et al, 2004; Silva & Romão, 2009). The retained EJC(s) can
interact with the translation termination complex via bridging interactions between
the release complex-associated proteins, UPF1 and SMG-1 (Kashima et al, 2005). This
bridging interaction has been proposed to trigger accelerated decay (i.e. NMD) of the
PTC-containing mRNA (Peixeiro et al, 2011).
I.2.2. Molecular events that prepare mRNA for degradation by
NMD
Translation termination is triggered by recognition of the stop codon by eRF1 and
eRF3. The central feature of the “unified model” of NMD is that the mechanism of
8
translation termination at a PTC is intrinsically different from translation termination at
a “correct” termination codon. In Saccharomyces cerevisiae, it has been demonstrated
that ribosomes do not efficiently dissociate from the mRNA when stalling at a PTC,
presumably because in that special environment they cannot receive the terminationstimulating signal from Poly(A)-binding protein (PABP). Likewise, in flies and human
cells, artificial tethering of PABP into close proximity of an otherwise NMD-triggering
PTC efficiently suppresses NMD. Based on reported biochemical interactions, this
model proposes that the decision of whether NMD is triggered relies on a competition
between UPF1 and PABP for binding to eRF3 bound to the terminating ribosome.
According to this model, a translation termination event is defined as “correct” if the
ribosome stalls close enough from the poly(A) tail to efficiently interact with PABP,
which, through yet unknown mechanisms, leads to a fast/efficient polypeptide release
and dissociation of the ribosomal subunits. If, in contrast, the spatial distance between
the terminating ribosome and the poly(A) tail is too big for this interaction to occur,
UPF1 will bind to the ribosome-bound eRF3 instead. Using co-immunoprecipitation
analysis, a recent study showed that UPF1, eRF1, and eRF3 are part of a single complex
that also contains the UPF1 kinase Suppressor with Morphogenetic effect on Genitalia1 (SMG1) (Kashima et al, 2006). This SMG1 - UPF1 - eRF1 - eRF3 (SURF) complex is
proposed to assemble on ribosomes stalled at a stop codon, with eRF1 and eRF3
recruiting unphosphorylated UPF1, which in turn recruits SMG1. At this stage, UPF1
might still be displaced by PABP and NMD would be prevented. However, when this
signal is absent, UPF2 and UPF3 will eventually bind UPF1, forming the decay-inducing
complex (DECID), which is required for SMG1 to phosphorylate UPF1. UPF1
phosphorylation is believed to definitively commit the mRNA for degradation by NMD,
maybe by inducing a conformational change that causes UPF1 to bind the mRNA.
Finally, the phosphorylated UPF1 will be bound by the 14-3-3-like phosphoserinebinding domains of SMG5, SMG6 and/or SMG7, ultimately leading to the degradation
of the mRNA. Phosphorylation of UPF1 triggers a critical step of translational
repression that is required before the messenger ribonucleoprotein particle (mRNP)
can be degraded. This step involves an interaction between phosphorylated UPF1 and
eIF3 that is part of the 43S ribosomal complex at the initiation codon of an NMD
target. This interaction inhibits 60S ribosomal subunit joining to form a translationally
9
active 80S ribosome and thus further translation initiation events on the mRNP (Chang
et al, 2007; Mühlemann et al, 2008; Schweingruber et al, 2013; Popp & Maquat, 2013).
Finally, the phosphorylated UPF1 will be bound by the 14-3-3-like phosphoserinebinding domains of SMG5, SMG6 and/or SMG7, ultimately leading to the degradation
of the mRNA (Mühlemann et al, 2008).
I.2.3. NMD degradation mechanism
Although good progress has been made in the understanding of the PTC-recognition
mechanism, little is known about the subsequent degradation of the recognized
nonsense mRNA. Current models propose that the factors SMG5, SMG6 and SMG7 are
involved in this process through SMG5/SMG7-mediated exonucleolysis and interaction
of SMG6 with phospho-UPF1 leads to a SMG6-mediated endonucleolytic cleavage near
the aberrant termination site (fig 4) (Nicholson & Mühlemann, 2010; Nicholson et al,
2010).
A common concept in metazoan NMD seems to be that phosphorylated UPF1 induces
various mRNA decay activities by recruiting decay factors or adaptor proteins for decay
complexes through its N- and C-terminal phosphorylation-sites (Schweingruber et al,
2013).
During NMD, mRNAs containing a phosphorylated UPF1 are committed to destruction.
Subsequent interaction of UPF1 with UPF2 and UPF3 directs SMG1-mediated
phosphorylation of UPF1, which in turn recruits SMG5, SMG7 and/or SMG6, which has
endonucleolytic activity in its PIN (PilT N-terminal) domain. Finally, this leads to SMG6mediated endocleavage near the PTC (Eberle et al, 2008). Irreversible endonucleolytic
cleavage by SMG6 generates a 5’ cleavage product that includes the PTC and a 3’
cleavage product that contains the EJC and NMD components. The 5’ cleavage product
is subject to 3’ to 5’ decay, possibly by the exosome, or to alternative decay pathways
involving degradation from either of the RNA termini (Popp & Maquat, 2013), such as
deadenylation-independent,
and
endonucleolytic
cleavage
dependent
decay
(Mühlemann et al, 2008).
10
Irreversible endonucleolytic cleavage by SMG6 generates a 5’ cleavage product that
includes the PTC and a 3’ cleavage product that contains the EJC and NMD
components. The 5’ cleavage product is subject to 3’ to 5’ decay, possibly by the
exosome. The 3’ cleavage product must meanwhile be stripped of its protein
components in order to be accessible to nucleases, and this is the job of UPF1. UPF1
activity is normally auto-inhibited by its own N- and C-terminal domains, but when
UPF1 binds to UPF2, it undergoes a large conformational change that activates its
helicase activity. UPF1 helicase activity disassembles proteins bound to the 3’ cleavage
product, recycling NMD factors and facilitating 5’ to 3’ exonucleolytic degradation by
the exoribonuclease XRN1 following 5’ cap removal by DCP2 initiated by the NMD
factors. Phosphorylated UPF1 also recruits SMG5, an adaptor that binds either prolinerich nuclear receptor 2 (PNRC2) or SMG7, each of which in turn recruits activities that
result in mRNA decapping followed by 5’ to 3’ degradation, deadenylation followed by
3’ to 5’ degradation, or both (Popp & Maquat, 2013; Nagarajan et al, 2013). This
pathway of deadenylation-dependent RNA decay represents the major decay
mechanisms for RNA-turnover in the cytoplasm (Nagarajan et al, 2013).
Research carried out in S. cerevisiae suggests that nonsense mutated mRNAs are
rapidly degraded using the main mRNA-turnover pathway as outlined above with a
modification thereof that is typified by deadenylation-independent decapping and
XRN1-mediated 5’ to 3’ exonucleolytic decay. In mammalian cells, studies have shown
that nonsense mutated mRNAs can be degraded via the conventional mRNA-turnover
pathway, starting with deadenylation, followed by decapping and XRN1-mediated
exonucleolytic decay (Nicholson & Mühlemann, 2010; Nicholson et al, 2010). Further
work is required to determine the relative contributions of the two decay pathways
involved in mammalian NMD and to understand what determines which decay route is
taken by the different types of mRNAs directed to the NMD pathway (Nicholson &
Mühlemann, 2010).
11
Figure 4: Model for degradation of NMD substrates. The model posits that UPF1-bound
mRNAs can be degraded by two different pathways, depending on whether the SMG-5/SMG-7
heterodimer or the endonuclease SMG-6 binds to phosphorylated UPF1. Interaction of SMG-5/
SMG-7 with phospho-UPF1 promotes deadenylation followed by decapping and exonucleolytic
RNA decay from both ends (left branch). Interaction of SMG-6 with phospho-UPF1 leads to a
SMG6-mediated endonucleolytic cleavage near the aberrant termination site, followed by the
exonucleolytic degradation of the two RNA fragments from the initial cleavage site (Nicholson
et al, 2010).
12
I.2.4. NMD targets
NMD is one of a number of mammalian-cell mRNA decay pathways that is coupled to
the translation of its target. Regulators of translation must be considered regulators of
NMD (Schoenberg & Maquat, 2012). The discovery that not only the initially identified
PTC-containing mRNAs but also many PTC-less mRNAs are targeted by NMD (re-)posed
the question, which features render an RNA susceptible to NMD and pointed out our
limited understanding of the mechanism of substrate selection (Schweingruber et al,
2013).
Until a few years ago, NMD has been seen merely as a quality control system that rids
the cell of faulty mRNAs, but recent studies indicate that NMD represents a much
more sophisticated tool serving multiple purposes in gene expression. The population
of NMD substrates is not only restricted to faulty transcripts, but also comprises
numerous endogenous, physiological transcripts. Among those are: i) mRNAs
containing short upstream ORFs (uORFs) were the termination codon of the uORF is
likely to be interpreted as PTC, unless the mRNA harbours stabilizing elements nearby;
ii) mRNAs encoding selenocysteine-containing proteins were UGA can be recognized as
codon for selenocysteine or as PTC, depending on endogenous selenium
concentration; iii) mRNAs harbouring introns in their 3’ UTR; iv) mRNAs with long
3’UTRs, which, in experimental situations generally make transcripts sensitive to NMD;
and v) transposons and retroviruses, likely due to indirect effects of NMD pathway
disruption. In addition, pseudo-genes, bicistronic mRNAs, and mRNAs containing
signals for programmed frameshifting have been identified as NMD targets in yeast.
NMD acts on them because their termination codons can be interpreted as a PTC
(Mühlemann et al, 2008; Chapin et al, 2014).
I.2.5. Implication of NMD in disease
Nonsense mutations account for ~11% of all gene lesions known to cause human
inherited disease and ~20% of disease-associated single-base pair substitutions
affecting gene coding regions (Mort et al, 2008).
13
There are numerous examples of human diseases associated with mutations that
result in PTCs. If translated, the PTC-containing mRNAs would give rise to truncated
proteins that have either completely lost their function, are still functional, have
acquired dominant-negative function or have gained new functions. As a consequence
of these different possibilities, NMD has a double-edged effect on the manifestation of
a disease: NMD can act either in a beneficial or in a detrimental way; the former if it
prevents the synthesis of toxic truncated proteins and the latter if it prevents the
production of proteins with some residual function. Thus, NMD represents a crucial
modulator of the clinical outcome of many genetic diseases (Nicholson et al, 2010;
Nicholson & Mühlemann, 2010).
There are many well-studied examples of human phenotypes resulting from nonsense
or frameshift mutations that are modulated by NMD. The phenotypes of genetic
diseases are likely to be frequently affected by NMD as PTCs are present in
approximately one-third of the human genetic disorders (Khajavi et al, 2006).
The majority of PTC-containing disease-associated alleles exert their negative effects
due to insufficient production of a functional protein. An example where NMD
aggravates the clinical outcome is provided by several disease phenotypes caused by
mutations in the dystrophin gene. While most of the truncating mutations in the
dystrophin gene are associated with a similar phenotype, the rare truncating
mutations that occur near the 3’ end of the dystrophin gene can result in extremely
variable phenotypes. It has been suggested that all truncated proteins encoded by
genes with mutations near the 3’ end would in theory be capable of rescuing the
Duchene’s Muscular Distrophy phenotype, but when NMD prevents their synthesis,
the clinical manifestations of the disease are aggravated. Conversely, NMD has a welldocumented beneficial role in the degradation of PTC-containing β-globin mRNA,
thereby preventing the synthesis of C-terminally truncated β-globin that would
otherwise cause toxic precipitation together with surplus α-globin chains. In a
heterozygote context, the second wild-type allele supports almost normal levels of βglobin synthesis, contributing to the correct haemoglobin assembly, which is reflected
in the recessive inheritance of this β-thalassaemia type. However, rare NMD-
14
insensitive PTCs are responsible for the dominant form of β-thalassaemia (Nicholson et
al, 2010).
I.2.6. β-Thalassaemia as a model disease for studying NMD
β-thalassaemia is one of the most common genetic diseases worldwide (Higgs et al,
2012). β-Thalassaemias are a heterogeneous group of inherited human anaemia’s
characterized by reduced or absent β-globin chain synthesis, resulting in reduced
haemoglobin in red blood cells, decreased red blood cells production and anaemia.
They are attributed to mutations within or upstream of the β-globin gene. The majority
of these mutations are frameshift or nonsense mutations, which are the most
prevalent β-globin mutations that cause β-thalassemia, within an exon that have no
effect on gene transcription or RNA splicing but result in the premature termination of
β-globin mRNA translation (Lim et al, 1989; Galanello & Origa, 2010; Peixeiro et al,
2011).
The major molecular consequences of stop mutations are the promotion of premature
translational termination and NMD. NMD will therefore prevent the production of
truncated and faulty proteins, which if failed could result in the synthesis of abnormal
proteins that can be toxic to cells through dominant-negative or gain-of-function
effects. This nonsense mediated mRNA decay has been found in bacterial, yeast, plant
and mammalian cells. It has been proposed that in the human β-globin gene,
mutations causing translation premature termination in exons 1 and 2 result in a
decrease of the mRNA from the affected allele, causing a 50% reduction of total βglobin chain synthesis in the heterozygote (Romão et al, 2000; Salvatori et al, 2009a).
In β39-thalassaemia the CAG (glutamine) codon of the β-globin mRNA is mutated to
the UAG stop codon, leading to premature translation termination and to mRNA
destabilization through the well-described NMD. Other examples of stop mutations of
the β-globin mRNA occur at positions 15, 37 and 127 of the mRNA (Salvatori et al,
2009a). In contrast, results obtained by Romão et al, 2000 from the study of nonsensemutated mRNA at codons 5, 15 or 17 indicate that the human β-globin mRNA carrying
a nonsense mutation in the 5’ half of exon 1 escapes NMD, the AUG-proximity effect.
15
On the other hand, if a PTC is localized in the last exon of the β-globin gene, the
transcripts produced are NMD-insensitive and are translated into truncated proteins
with dominant negative effects, as is the case of the stop mutation at position 127
(Mühlemann et al, 2008; Salvatori et al, 2009a).
The fact that nonsense mutations promote premature translational termination and
are the leading cause of up to 30% of inherited diseases, one of them being
thalassemia, and given the small size of the β-globin gene and the wide range of
nonsense mutations that have been described at this locus make this disease an
attractive model for investigating the effects of premature translation termination on
mRNA metabolism (Salvatori et al, 2009b; Romão et al, 2000).
I.2.7. Suppression therapy
For many genetic disorders caused by PTC-generating mutations, there are no effective
treatments available. Because NMD plays an important role in modulating the clinical
manifestations of such diseases, interfering with NMD represents a promising
therapeutic strategy. For those cases where the truncated protein is still functional,
inhibiting rapid degradation of the nonsense mRNA would in principle suffice to
elevate the protein concentration and ameliorate the condition of patients. However,
in most cases, production of the full-length protein would be necessary to restore
function, which can be achieved by promoting readthrough of the PTC (Mühlemann et
al, 2008). When a stop codon enters the ribosomal A-site, the sampling process is
initiated just as it does at a sense codon. Near-cognate aminoacyl tRNAs with
anticodons that are complementary to two of the three nucleotides of a stop codon
can compete with the release factors for A-site binding. Normally, stop codon
recognition by the eRF1/3 complex efficiently out-competes near-cognate aminoacyl
tRNAs and efficient polypeptide chain release occurs. On occasion, aminoacyl tRNAs
that are near-cognate to a stop codon become accommodated in the ribosomal A-site
and their amino acid is incorporated into the polypeptide. This process that recodes a
stop codon into a sense codon is referred to as a “readthrough” event. PTC
readthrough suppresses translation termination and allows translation elongation to
continue in the correct reading frame until the normal stop codon is encountered
16
(Keeling & Bedwell, 2011). Given that the presence of PTCs codons explain one third of
all described inherited human diseases (Bhuvanagiri et al, 2010), therapeutic strategies
aimed at suppressing nonsense codons (so-called nonsense suppression therapies)
have the potential to provide a therapeutic benefit for patients with a broad range of
genetic diseases (Keeling et al, 2014; Keeling & Bedwell, 2011).
The goal of suppression therapy is to enhance the ability of near-cognate aminoacyl
tRNAs to compete with the release factor complex for binding PTCs in the ribosomal A
site. By increasing the frequency that PTCs are recoded into sense codons, enough full
length, functional protein may be restored to provide a therapeutic benefit to patients
that carry PTC containing transcripts (Keeling & Bedwell, 2011). In the last few years, it
has been demonstrated that drugs can be designed and produced to suppress
premature termination, inducing a ribosomal readthrough of PTCs in eukaryotic
mRNAs. In order to develop an efficient suppression therapy, aminoglycoside
antibiotics, including gentamicin, amikacin, paromomycin,
geneticin
(G418),
lividomycin, tobramycin, and streptomycin have been tested on mRNAs carrying PTCs
and shown to suppress disease-causing PTCs in mammalian cells. These drugs bind the
decoding centre of the ribosome and decrease the accuracy in the codon-anticodon
base-pairing, inducing a ribosomal readthrough of premature termination codons
partially restoring protein function to various extents for more than twenty different
disease models in vitro, and eight different disease models in vivo (Mühlemann et al,
2008; Salvatori et al, 2009a; Salvatori et al, 2009b; Keeling & Bedwell, 2011).
However, though these results are promising there are still several obstacles that must
be overcome before aminoglycosides can be used long-term in the suppression of
nonsense mutations. First, the efficiency of suppressing PTCs is greatly influenced by
the identity of the stop codon and the surrounding mRNA sequence. Various
aminoglycosides have different abilities to suppress PTCs. This suggests that screening
compounds to identify those that best suppress a particular PTC in its natural sequence
context is needed. Second, the long-term use of aminoglycosides is limited due to side
effects and not all patients respond in the same way to these drugs which could
possibly be due to the different efficiencies of NMD, from individual to individual
(Keeling & Bedwell, 2011; Welch et al, 2007).
17
I.3. Aims
It has been estimated that about 1.5% of the global population (80 to 90 million
people) are carriers of β-thalassemia, with about 60,000 symptomatic individuals born
annually, the great majority in the developing world (Galanello & Origa, 2010). The
majority of the mutations associated with β-thalassemia are nonsense mutations
which result in the premature termination of β-globin mRNA translation and
consequently NMD (Galanello & Origa, 2010; Peixeiro et al, 2011). Therefore, it would
be of great interest that these nonsense mutations in the β-globin gene could be
suppressed with the use of drugs such as aminoglycosides. Preliminary results
obtained in our lab had shown that the aminoglycoside G418 can suppress a PTC at
codon 39 of the human β-globin mRNA, although at low levels in cultured erythroid
cells. The aim of the work carried out in this thesis was to further prove that
suppression therapy can restore enough β-globin protein and therefore correct the
disease manifestations of β-thalassemia. In this regard, it was decided to test whether
G418 is able to induce efficient levels of suppression in a dose-dependent and timecourse manner in HeLa cells transfected with plasmids containing the wild type (βWT)
or β39 human β-globin genes.
II. Methods
II.1 Plasmid constructs
The plasmids containing βWT (wild type version of the β-globin gene), β15 (with
mutation at codon 15) [CD 15 (TGG→TGA)], or β39 (with mutation at codon 39) [CD 39
(CAG→TAG)] human β-globin gene were obtained as previously described in Romão et
al, 2000. All variants were created within the 428-bp NcoI-BamHI fragment of the βglobin gene template by overlap-extension PCR. Competent Escherichia coli were
transformed with the plasmid DNA, and transformants were selected on luria-bertani
(LB) agar/ampicillin plates. The corresponding plasmid DNAs were purified from
overnight cultures of single colonies with the NZYMini prep kit (NZYTech, Portugal)
following the manufacturer’s instructions. Confirmation of the correct cloned
sequences containing the relevant mutation was carried out by automatic sequencing.
18
II.2 Cell culture and plasmid transfections
HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM 1x +
GlutaMAXTM-I; Gibco® by Life Technologies™, USA) supplemented with 10% (v/v)
foetal bovine serum (FBS; Gibco® by Life Technologies™, USA), incubated at 37°C in a
humidified atmosphere of 5% CO2.
Transient transfections were performed using Lipofectamine 2000 Transfection
Reagent (Invitrogen® by Life Technologies™, USA), following the manufacturer’s
instructions, in 35-mm plates containing HeLa cells plated 24h prior to transfection,
using 400 ng, of plasmid DNA of each variant (βWT, β15 and β39). Twenty-four hours
post-transfection, cells were either treated or untreated with G418 (Sigma-Aldrich®,
USA). The culture medium was removed and new medium supplemented with 0
µg/ml, 50 µg/ml or 200 µg/ml of G418 (Sigma-Aldrich®, USA) was added. The medium
was not changed during the treatment period. Cells were harvested 12h and 24h post
treatment with the above mentioned drug by rinsing twice with Phosphate Bufferd
Saline (PBS) and lysed via solubilisation in Passive Lysis Buffer (PLB; Promega, USA).
II.3 RNA isolation
Total RNA from cultured HeLa cells was isolated using the RNA extraction kit
NucleoSpin RNA II (Macherey-Nagel, Germany) according to the manufacturer’s
instructions. RNA samples were treated with RNase-free DNase I (Ambion® by Life
Technologies™, USA) and purified by phenol-chloroform extraction.
II.4 Reverse transcription-coupled quantitative PCR (RT-qPCR)
cDNA synthesis was carried out using 2 μg of total RNA and Reverse Transcriptase
(NZYTech, Portugal), according to the manufacturer’s instructions. Real-Time
quantitative PCR (RT-qPCR) was performed in ABI Prism 7000 Sequence Detection
System, using SybrGreen Master Mix (Applied Biosystems® by Life Technologies™,
USA). Primers specific for the gene of interest, β-globin (primer forward 5’GTGGATCCTGAGAACTTCAGGCT-3’ and primer reverse 5’-CAGCACACAGACCAGCACGT3’) and for the control, puromycin resistance gene (primer forward 5’19
GGGTCACCGAGCTGCAAGAA-3’ and primer reverse 5’-CACACCTTGCCGATGTCGAG-3’)
were designed. Quantification was performed using the relative standard curve
method (ΔΔCt, Applied Biosystems® by Life Technologies™, USA). The following cycling
parameters were used: 10 min at 25°C and then 50°C for 30 min and 5 min at 85°C.
Technical triplicates from each experiment were assessed in all cases.
II.5. SDS-PAGE and Western blotting
Cells lysates were denatured for 10 minutes at 65°C. Five µl of SDS sample buffer 5x
[Bromophenol blue (0.25%), DTT (dithiothreitol; 0.5 M), Glycerol (50%), SDS (sodium
dodecyl sulfate; 10%), Tris-Cl (0.25 M, pH 6.8)] was added to 20 µl of purified lysates
and these were loaded in to a 12% polyacrylamide gel and resolved for 1 hour. After,
they were transferred to a PVDF membrane (Bio-Rad, USA) for 1 hour. The membrane
was blocked in 5% (w/v) nonfat dry milk for 1 hour and probed using mouse anti-αtubulin antibody (loading control; Roche, Switzerland) at 1:10 000 dilution and mouse
monoclonal anti-β-globin (Santa Cruz Biotechnology, USA) at 1:200 overnight. After
incubation with the primary antibody, membranes were washed 3 times in TBS-Tween
20 (Sigma-Aldrich®, USA) 0.05% (v/v) and 0.1% (v/v). Detection was carried out by
incubating the membranes for 1 hour with the secondary antibodies, peroxidiseconjugated anti-mouse IgG (Bio-Rad, USA), anti-rabbit IgG (Bio-Rad, USA) antibodies,
followed by enhanced chemiluminescence reaction.
Seen as we were unable to detect protein, optimizations had to be made to the
protocol. We first altered the dilution of the mouse monoclonal anti-β-globin, the
concentration of TBS-Tween 20 was lowered, the washing times were also altered and
finally the exposure times were increased. We will further discuss these alterations in
point III.2.2.
II.6. Statistical analysis
Results are expressed as mean ± standard deviation of 3 experiments in which the
mRNA levels expressed from β15 and β39-containing plasmids are normalized to the
wild type mRNA levels arbitrarily set to 1. Student’s t test was used for estimation of
20
statistical significance (unpaired, two tails). Significance for statistical analysis was
defined as a p< 0.05 (Livak & Schmittgen, 2001).
III. Results and Discussion
III.1. Sequence analysis of the plasmid DNAs encoding the β-globin
gene
The aim of this study was to prove that suppression therapy by treatment with G418
can restore correct β-globin protein. For this purpose, we chose to transiently express
a plasmid encoding the β-globin gene in HeLa cells and check the levels of expression
of this gene in the presence or absence of a drug that promotes readthrough of a PTC.
For that, we used, alongside the wild type version of the gene, two other versions: one
with a nonsense-mutation in codon 15, which is imminent to NMD, due to the AUGproximity effect (Silva et al, 2008); and one version of the gene carrying a nonsense
mutation in codon 39, which is typically committed to NMD (Romão et al, 2000). In
order to check if we were using the plasmids expressing the correct versions of the
gene, we sequenced the previously obtained plasmids (Romão et al, 2000) to confirm
the presence of the mutation in codon 15 and 39 and the correct sequence in wildtype. Sequence analysis of the amplified plasmids encoding the β-globin gene (wildtype, with mutation at codon 15 and with mutation at codon 39) confirmed the
presence of a mutation at codon 15 (TGG→TGA) or 39 (CAG→TAG) (fig 5). In addition,
sequence analysis of the βWT gene did not reveal any mutation (fig. 5).
A
βWT
β15
21
B
βWT
β39
Figure5: Sequencing analysis confirmed the presence of nonsense mutations that originated
a stop codon at codons 15 and 39 of the β-globin gene, respectively. The chromatogram of
the amplified wild type β-globin gene-containing plasmid DNA is shown on the left in order to
compare to the sequencing reaction of the amplified plasmid DNA containing the β15 gene (A)
shown on the right and the β39 gene (B) shown also on the right. Underlined sequences
indicate codon 15 (A) and codon 39 (B). N indicates that the algorithm is unable to identify the
base. Even though this happens, due to a very low cut-off, it is still possible to identify which
base is because the peek is very clear.
III.2. Analysis of βWT, β15 and β39 transcripts in transiently
transfected HeLa cells
As mentioned above, it has previously been shown that transcripts carrying mutations
in exon 1 of the human β-globin gene (namely at codon 15) are expressed at levels
approaching those of the wild-type β-globin mRNA in erythroid cells, which means that
this AUG-proximal nonsense-mutated gene escapes NMD (Silva et al, 2008). On the
other hand, those at positions downstream of codon 24, such as β39 transcripts, are
expressed at low levels indicating that they undergo fully efficient decay (Romão et al,
2000). As these mutations have been proven to have β-thalassaemia clinical relevance
we analysed whether these mutations causing premature translation termination
could be suppressed in HeLa cells with the use of aminoglycosides. For that, we
transiently transfected HeLa cells with the plasmids carrying the βWT, β15 or β39
genes. Twenty-four hours later cells were harvested and protein and RNA were
isolated for analysis.
22
III.2.1. mRNA quantification by RT-qPCR analysis
The relative mRNA levels were quantified by RT-qPCR and normalized to the level of
the βWT mRNA (arbitrarily set to 100%) (fig 6). Results show that β15 relative mRNA
level is 70% of the βWT mRNA. In contrast, the level of β39 mRNA indicates that rapid
decay has occurred: 31% of βWT mRNA.
Transfected constructs
Figure 6: RT-qPCR analysis confirms that the transcript containing the mutation at codon 39
is degraded via NMD. (A) Representative RT-qPCR analysis of RNA isolated from untreated
HeLa cells transiently transfected with the constructs specified beneath each lane. Resulting
mRNA levels were normalized to the expression level of the wild-type mRNA. 0 represents
non-transfected HeLa cells which serves as negative control of the transfection efficiency as
these cells are not able to express the β-globin gene endogenously. C-βWT corresponds to the
reverse transcription reaction without the reverse transcriptase enzyme which serves as a
control to detect the presence of DNA contamination.
These data were expected as they confirm what was previously reported by Romão et
al, 2000 and Inácio et al, 2004 revealing that the tested β15 transcript with a PTC
located upstream of codon 24 is able to escape NMD, producing mRNA levels similar to
that of βWT, whereas the transcript mutated at a position downstream of codon 24, as
is the case with β39, undergoes rapid decay through NMD.
23
III.2.2. Protein analysis by Western Blot
Western blot analysis of protein samples extracted from the cells transfected with
each one of the plasmids was performed in order to evaluate protein levels produced
by each transcript (fig 7). As one can observe in figure 5, as far as protein production is
concerned, it was unable to detect any production of β-globin protein. This may be as
a result of low signal visibility, consequence of low concentration of the antibody or
antigen or prolonged washing, and the buffers used may also contribute to the
problem. Therefore the conditions in which the Western blot was performed were
optimized. Primary antibody dilution was altered from 1:200 to 1:100, increasing its
concentration. Incubation time with the primary antibody was increased to intensify
the signal. Even though β-globin is not a phosphorylated protein, Bovine Serum
Albumine (BSA) was used to see if it produces any significant alteration in the
detection of the protein. The stringency of the washing conditions was decreased.
Finally, exposure times were increased from 1 s, 30 s, 1 min and 2 min, to 1 s, 1 min, 5
min, 10 min, to achieve an optimum time. After various alterations to our initial
protocol (based on manufacturer’s instructions) Western blot analysis was performed,
yet different conditions were used, we were still unable to detect protein. It was
hypothesized that this may be because the conditions in which the cells were kept may
somehow cause them some stress inhibiting translation. However, this is not the case
since it was able to detect α-tubulin, the loading control, a protein that is produced
endogenously. If cells can produce this protein, the translation machinery is working.
Also, α-tubulin is cap-dependent translated, so, a priori, no translation factor is
missing. It was also hypothesized that the antibody may have not been produced
properly. The ideal at this point would be to try a new antibody and if this wouldn’t
work to try the antibody on a different cell line that produces the β-globin gene
endogenously. Due to time and money contingencies, this was not feasible and
therefore it was decided to continue the work studying the effect of the drug of choice
(G418) only on the mRNA, while trying to understand why the antibody was unable to
detect protein. This may possibly indicate some lines of evidence on the effect of the
drug at mRNA level and produce some more knowledge regarding this matter.
24
βWT
β15
β39
0
α-tubulin (55kDa)
β-globin (16 kDa)
Figure 7: Western blot analysis of cell lysates. The molecular weights of the α-tubulin (55 kDa)
and β-globin (16 kDa) proteins are indicated on the right hand side of the autoradiograph. The
very faint bands that can be seen in βWT and β39 are artifacts.
III.3. Analysis of βWT and β39 transcripts in transiently
transfected HeLa cells exposed to geneticin (G418)
We transfected HeLa cells with plasmids expressing βWT and β39 genes. Twenty-four
hours later, cell cultures were either treated or untreated with increasing
concentrations (50 µg/ml or 200 µg/ml) of G418 and harvested 12 or 24 hours later.
The β-globin mRNA levels were quantified by RT-qPCR analysis (fig 8). Results have
shown that the relative mRNA level of the β39 transcript is approximately 40% of the
βWT mRNA, which is arbitrarily set to 100% (fig.8A). These data indicate that these
nonsense-mutated mRNAs undergo rapid decay, as expected. On the other hand β39
mRNA levels of G418 treated cells is at similar levels (fig. 8B and C), although there is
an increase
A
25
B
C
Figure 8: RT-qPCR analysis performed on RNA isolated from HeLa cells containing the βWT
and β39 transcripts treated with increasing doses of G418 in a time-course manner. Panel A
depicts an untreated control as cells were lysed 24-hours post-transfection and were not
treated with any amount of G418. Resulting levels of relative mRNA levels are normalized to
the expression level of the wild-type mRNA. Panels B and C depict treated cells with the
indicated amounts of G418 for 12 hours and 24 hours, respectively.
of 15% in β39 mRNA levels in the presence of 50 µg/ml of G418 and an increase of 25%
in the presence of 200µg/ml of G418, compared to no treatment conditions (fig. 8 B);
26
at 24h, there is no change in mRNA levels in the presence of 50 µg/ml of G418,
whereas in the presence of 200µg/ml of G418 it is possible to see an increase of 20%
compared to control conditions (fig. 8 C). Although we observe an increase in β39
mRNA levels upon treatment unfortunately it is not significant. The great standard
deviations are due to the fact that the experiments all resulted in very different CT
values. The observed increase may be due to the ability of the aminoglycoside to bind
to the decoding centre of the ribosome leading to the reduction of translation fidelity
by reducing the proofreading ability of the ribosome and increasing the
misincorporation of near-cognate aminoacyl tRNAs into the ribosomal A site at stop
codons, resulting in translational misreading at PTCs without premature termination of
protein synthesis with concomitant NMD inhibition. However one can not be sure
about this seen as it is not possible to see the effects of the drug on protein synthesis.
Another possibility could be the ability of the drug to reduce the sensitivity of the NMD
machinery to the presence of a PTC, which would be a side-effect of the drug. However
it has been shown that this aminoglycoside, and various others, is capable of
suppressing disease-causing PTCs in mammalian cells expressing genes carrying these
mutations, without altering global protein or mRNA profiles (Keeling & Bedwell, 2011).
IV.
Future directions
While the results regarding the steady increase of mRNA levels after treatment with
G418 are promising, a previous report describes a promising drug, PTC124 by PTC
Therapeutics, Inc., which is able to suppress nonsense mutations by a readthrough
activity, as well as, or better than aminoglycosides, at nanomolar concentrations in
mammalian cells without toxic side effects. This molecule is administered orally and is
expected to be very promising in therapy. PTC124 has no structural similarity to
aminoglycosides or other clinically developed drugs and was found to be safer offering
therapeutic benefits to many patients (Keeling & Bedwell, 2011; Welch et al, 2007;
Salvatori et al, 2009a). Given that this compound is currently undergoing clinical trials
for several diseases (Keeling & Bedwell, 2011) and that thalassemia is a major health
problem in developing countries (Salvatori et al, 2009a; Galanello & Orita, 2010), it
27
would be of great interest to test this compound in its ability to suppress PTCs in the βglobin gene in comparison with the effects of G418. It has also been shown that
tethering PABPC1 in close proximity of a NMD-competent PTC inhibits NMD (Silva et al,
2008; Behm-Ansmant et al, 2007). Based on these data we suggest testing if we could
increase the efficiency of suppression therapy by transfecting cells with antisense
oligonucleotides specific for the β-globin mRNA sequence but with a degenerated
poly(A) tail in combination with one of the drugs tested. Each oligonucleotide must
have a poly(A) tail long enough to bind at least two PABPC1 molecules which will allow
PABPC1 to bind and thus to inhibit NMD. The combination of these two treatments
may give a promising and efficient therapy.
28
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